Preoperative differentiation of benign and malignant/potentially malignant pancreatic cystic lesions is problematic. Data to support the role of EUS and EUS-guided fine-needle aspiration (EUS-FNA) are limited. This study assessed the sensitivity, specificity, and accuracy of EUS, cytopathology, and analysis of cyst fluid for pancreatic cystic lesions. Retrospectively, 111 consecutive patients were identified (54 men, 57 women; mean age 59 years, range 18-79 years) who underwent EUS from July 1997 to September 2000 because of known or suspected pancreatic cystic lesions based on CT or transabdominal US. Thirty-four patients (16 men, 18 women; mean age 55 years, 25-79 years) who underwent surgery formed the basis for this analysis. EUS diagnosis was compared with surgical pathology. Selected patients underwent EUS-FNA to obtain specimens for cytopathologic analysis and for determination of carcinoembryonic antigen levels. Based on surgical pathology, cysts were classified as benign (simple cyst, pseudocyst, serous cystadenoma) or malignant/potentially malignant (mucinous cystadenoma, intraductal papillary mucinous tumor, cystic islet cell tumor, cystic adenocarcinoma). EUS-FNA with cytopathologic assessment of cyst fluid was performed for 18 of the 34 patients; carcinoembryonic antigen level was determined in 11 cases. For EUS, cytopathology, and carcinoembryonic antigen, sensitivity was, respectively, 91%, (p = 0.01 vs. cytology), 27%, and 28%; specificity was, respectively, 60%, 100%, and 25%; and, accuracy was, respectively, 82%, 55%, and 27%. The sensitivity of EUS in all 13 patients with cystic islet cell tumor, intraductal papillary mucinous tumor, or cystic adenocarcinoma was 100%. Combining EUS, cytopathology, and carcinoembryonic antigen results did not improve accuracy. There were no complications related to the EUS or EUS-FNA. EUS alone is sensitive and accurate in identifying malignant/potentially malignant pancreatic cystic lesions. EUS-FNA to obtain specimens for cytopathologic analysis and determination of carcinoembryonic antigen levels, although safe, does not enhance diagnostic yield.